Flow cytometry によるBromodeoxyuridine(BrdU) /DNA同時解析法の応用 1.その基礎的・技術的検討

Abstract

FCMによるBrdU/DNA同時解析法の臨床応用を目的として, その基礎的・技術的検討を行った.1) In vitroにおけるBrdU標識のための至適条件は, 最終濃度10 μMのBrdU含有培養液中で30分間培養標識することであった.2) In vivoでのBrdU標識のための至適条件は, 40 μg/gマウス体重のBrdUを腹腔内1回投与し, 1時間標識することであった.3) DNA変性のための至適条件は, 4N塩酸を用い, 20°Cで20分間の変性処理を行うことであった.4)これらの条件下では, ほぼ満足のいくBrdU/DNA同時解析結果が得られた

The precise in vitro and in vivo BrdU labeling methods and the optimal combination of HCl concentration, treatment time and temperature for DNA denaturation for use in simultaneous flow cytometric BrdU/DNA analysis were investigated. An EPICS C Cell Sorter with computer analyser was used. 1) For precise labeling of exponentially growing MBT-2 cells the cell should be exposed to 10 microM BrdU for 30 min. 2) For optimal labeling of mouse subcutaneous MBT-2 tumor cells 40 micrograms of BrdU per gram should be injected intraperitoneally for 1 hour. 3) The procedure yielding the most quantitative results for DNA denaturation was treatment of the cells with 4N HCl for 20 min at 20 degrees C. Under these conditions, reliable data were always obtained. In the future, this flow cytometric two-color analysis will be applied to clinical fields and further development is expected.